Abnormal handling of intracellular Ca has been identified as a major cause of heart failure (HF), a leading cause of death in the US. Despite intense effort, the precise mechanisms underlying the HF-related alterations of Ca signaling remain poorly understood. The overall goal of this proposal is to define the role of alterations of the sarcoplasmic reticulum (SR) Ca release channel/ryanodine receptor (RyR) in HF. In cardiac muscle, RyRs are activated by cytosolic Ca via the mechanism of Ca-induced Ca release (CICR). Data obtained recently in our laboratory shows that in addition to cytosolic Ca, RyRs are also controlled by Ca inside the SR at luminal sites. The presence of a luminal Ca sensor regulating release of SR Ca has profound implications for our understanding of intracellular Ca signaling in normal and diseased myocardium. For example, by linking the functional activity of RyR channels to the loading state of SR, the luminal Ca sensor stabilizes Ca release in the face of alterations of RyRs. Consequently, any factors affecting the properties of RyRs are compensated by changes in SR Ca load and concomitant luminal Ca-dependent changes in RyR channel activity. Our preliminary data suggest that this feedback mechanism might be compromised in HF, potentially leading to chronic depression of SR Ca release function. In the present proposal we will address the following questions: 1) Are the intrinsic gating properties of RyRs (i.e. regulation by cytosolic and luminal Ca) altered in HF? 2) What are the precise molecular mechanisms of these alterations? And 3) How do the proposed changes in RyRs contribute to abnormal Ca cycling in HF? To this end, single channel approaches in a reconstitution system and fluorescent calcium imaging techniques in isolated ventricular myocytes and whole beating hearts will be utilized to determine relationships between RyR properties and HF at multiple levels, using several well-established animal models of HF. The results of this study will improve our understanding of the mechanisms and role of altered Ca handling in HF.